Poster Session (free drinks) 20:30 - 22:00

Conticelli D.^1,2, Migliore C.^1,2, Picco G.³, Calabrò E.¹, Bellomo S.E.^1,2, Maina I.M.^1,2, Orrù C.^1,2, Ribisi S.^1,2, Corso S.^1,2, Giordano S.^1,2

¹ University of Turin, Dept. of Oncology, Italy

² Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy

³ Wellcome Sanger Institute, Cambridge, UK

Introduction 

Around 22% of gastric cancer (GC) cases are characterized by microsatellite instability (MSI), a hypermutability condition caused by the impairment of the DNA mismatch repair (MMR) system. Inactivation of MLH1 or MSH2 genes are the main mechanisms leading to MMR deficiency in both familiar and sporadic MSI GCs. Compared to the other GC molecular subtypes, MSI tumors display a more favorable prognosis, a lower relapse frequency and a better response to immune checkpoint blockade. However, several studies reported the existence, among hypermutated cases, of two populations with different prognosis. Consistently, according to clinical studies testing the anti-PD1 antibody Pembrolizumab alone or in combination with chemotherapy, objective response rates in MSI GCs were close to 60%, suggesting that a significant fraction of tumors are intrinsically resistant. It is thus necessary to get more insights into MSI GC evolution, in order to properly stratify patients, maximize therapy efficacy and identify new cancer vulnerabilities to be exploited.

Material and Methods

To model MSI GC evolution, we established organoid cultures from the normal stomach mucosa of NOD/SCID and BALB/c mice. In line with some reports identifying the MSI status as an early event in gastric carcinogenesis, we generated MMR-deficient (MMRD) organoids using CRISPR/Cas9 technology to knock out MLH1 or MSH2 genes. We maintained both wild type (WT) and MMRD models continuously in culture for several months, evaluated their microsatellite status and molecular features over time, and tested their ability to develop tumor masses upon subcutaneous injection in mice.

Results and Discussion

Our models recapitulate gastric epithelial tissue as they express markers of stem and differentiated gastric cells (i.e. LGR5, MUC5AC, PGC). As expected, after 3 months in culture, MMRD -but not WT models- began to acquire MSI features according to microsatellite marker analysis. Upon subcutaneous injection in NOD/SCID mice, MMRD cells were able to generate tumors in two months. Retrieved cells did not express either Mlh1 or Msh2 and displayed a pronounced MSI phenotype. The evaluation of the Tumor Mutational Burden at several time points is ongoing, as well as the reconstruction of phylogenetic trees recapitulating the evolution of clonal subpopulations. We are also optimizing the model in order to obtain less-immunogenic (non-Cas9 expressing) MMRD BALB/c organoid clones suitable for injection in immunocompetent syngeneic mice to unravel the impact of the immune system and treatment with immune checkpoint inhibitors on tumor evolution.

Conclusion

We used an unbiased approach based on the inactivation of a single MMR gene to generate a powerful model of MSI GC suitable to study tumor evolution, heterogeneity and response to immunotherapy.

Alexandra Ambra Ulla², Consalvo Petti², Roberta Porporato², Ivan Molineris³, Elena Grassi^1,2, Ymera Pignochino^2,4, Andrea Bertotti^1,2, Alberto Bardelli^1,2, Enzo Medico^1,2, Claudio Isella^1,2

¹ Department of Oncology, University of Turin, Candiolo (TO), S.P. 142, km 3.95, 10060, Italy

² Candiolo Cancer Institute, FPO-IRCCS, Candiolo (TO), S.P. 142, km 3.95, 10060, Italy

³ Department of Life Sciences and Systems Biology

⁴ Department of clinical and biological sciences, University of Turin

Introduction

Recent studies revealed that an aggressive subgroup of colorectal cancer (CRC) is characterized by a distinct tumor growth pattern, defined by the high content of cancer-associated fibroblasts (CAF). However, both cells-intrinsic traits underpinning such phenotype and the characterization of CAF associated with poor prognosis is still missing.

Methods

To tackle these issues, we generated a biobank of CRC cell line xenografts (CLX), where normal mouse stroma cells are strictly educated from epithelial cancer cells.

Results

In vivo CLX showed high variability in growth rate, revealing a subgroup of slow-paced tumors, in which the epithelial cells display EMT-traits (EMRM) and concur with specific CAF population (mrCAF) in the remodeling of the extracellular matrix. In CRC scRNAseq data and pan-cancer study, EMRM and mrCAF matched traits can be detected across different tumor types, predicting poor prognosis.

Conclusion

Altogether, our results highlight the intrinsic capability of cancer cells to co-work with CAF, indicating route toward tumor progression involving the interaction between tumor and its microenvironment.

Jamal Elhasnaoui¹, Riccardo Corsi³, Elena Grassi¹, Ales Krenek², Andrea Bertotti¹, Luca Vezzadini³, Claudio Isella¹, Enzo Medico¹

¹ Department of Oncology, University of Torino, 10060 Candiolo, Italy

² Institute of Computer Science, Masaryk University, Šumavská 15, 60200, Brno, Czech Republic

³ Kairos3D S.r.l., 10100 Torino, Italy

Introduction

In cancer genomics, integrative analysis of data obtained from biological samples of patient cohorts requires handling large groups of patients, each with molecular data of different type for thousands of genes, such as expression, mutation, copy number, and others.

Currently available tools allow visualization and analysis of such complex data, however forcing the user to work with partial views due to the amount of information. Also, these systems usually involve somewhat laborious procedures, so that for a complete analysis of the information it is often necessary to use many tools in sequence.

Results and Discussion

Here we present GenomeCruzer, a software for 3D interactive visualization and analysis of multidimensional omic data sets (e.g. gene expression, methylation, copy number alteration). GenomeCruzer is particularly useful to quickly achieve clinical and biological insight, providing easy integration and interactive visualization of genomic data of any kind. GenomeCruzer has the dual objectives of representing large amounts of genomic data in an easily readable way, allowing interactive analysis and providing visual results in real-time. Powerful graphics also allow simultaneous display of different types of values for the same objects, through multiple dimensions and customizable visual metaphors. The user can navigate this 3D environment and interact with the different elements, which constitute a representation of the starting datasets, building analysis paths and using statistical tools e.g. for the comparison of groups to highlight correlations.

Conclusion

In conclusion, GenomeCruzer enables users without bioinformatics background to browse, analyse and interpret complex omics datasets for multiple groups of patients and genes simultaneously, extracting clinical correlates and biological knowledge.

Elisabetta Puliga¹, Tania Isabel Capeloa^2*, Claudia Orrù^1,2, Salvatore Ribisi^1,2, Daniela Conticelli^1,2, Irene Maina^1,2, Simona Corso^1,2, Silvia Giordano^1,2

¹ Candiolo Cancer Institute, FPO IRCCS, Candiolo, Italy

² University of Torino, Department of Oncology, Candiolo, Italy

^* Current Affiliation: Pole of Pharmacology, Institut de Recherche Expérimentale et Clinique (IREC), Universitè catholique de Louvain (UCL), Brussels, Belgium

Introduction

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide and the overall clinical outcome for patients with advanced disease is poor. Since the oncogenic role of KRAS mutations has been poorly studied in GC, taking advantage of a proprietary, molecularly annotated colony of GC PDXs we aim to i) molecularly and biochemically characterize different KRAS mutated models and ii) unravel KRAS mutants’ differences in terms of response to therapy.

Material and Methods

To address our analysis, we took advantage of a multi-level platform of GC models, comprising more than 211 PDXs, primary cell lines and organoids. Whole genome molecular analysis identified the presence of KRAS mutations in 19 PDXs (32% G12D and 21% A146T). Primary cell lines were established from G12D and A146T mutated PDXs and tested by Sanger sequencing to confirm the presence of KRAS mutations.

Results and Discussion

To characterize GC KRAS mutated models, we firstly evaluated the activation status of KRAS performing a RAS-GTP G-LISA. Models bearing the canonical mutation G12D presented higher levels of RAS activation compared to the models bearing the rare (but enriched in our platform) A146T mutation. These results are in according with the impact of different amino acid substitutions on the biochemical activity of KRAS. To investigate whether KRAS can be targeted in our mutated models, we evaluated cell addiction to this oncogene. Regardless the type of mutation, silencing experiments showed that mutated cells significantly rely on KRAS activity for survival. Since a direct targeting of G12D and A146T KRAS mutants is not a clinical option, we tested the sensitivity of these models to the MEK-inhibitor Trametinib alone or in combination with the AKT inhibitor MK-2206. While models bearing the G12D mutation benefited of the drugs combination, a strong effect of Trametinib alone was observed in 2 of the A146T mutated models analysed, suggesting a remarkable vulnerability for this mutant. The other two A146T mutated models showed a better response to the MK-2206 alone or to the combo likely because of the additional presence of point mutations in PI3K and PTEN genes respectively. These results were confirmed by biochemical analysis suggesting the possible involvement of pAKT in response to the MEK inhibition in canonical and in A146T+PI3K/PTEN mutated models but not in A146T mutants alone. This evidence was confirmed by in vivo studies showing the efficacy of the combo treatment in G12D and A146T+PI3K PDXs and the best efficacy of Trametinib alone in the A146T mutated model in the absence of additional driver mutations.

Conclusion

Together, these results highlight the importance of a better characterization of the different KRAS mutants. Indeed, a KRAS allele-specific therapeutic approach could be a better option to target KRAS in GC.

Marocchi F.¹, Palluzzi F.², Nicoli P.¹, Meliksetian M.¹, Bertalot G.³, Amati B.¹, Ferrucci P.F.¹, Bossi D.⁴, Lanfrancone L.¹

¹ Department of Experimental Oncology, European Institute of Oncology IRCCS, Milan, Italy

² Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Roma, Italy

³ Ospedale Santa Chiara di Trento, Trento, Italy

⁴ Institute of Oncology Research IOR – Functional Cancer Genomics, Bellinzona, Switzerland

Introduction

The introduction of systemic targeted therapies (BRAF and MEK inhibitors) and immunotherapy (CTLA-4 and PDL-1 immune checkpoint inhibitors) dramatically improved melanoma patients overall survival rates. Nevertheless, the majority of patients commonly experience recurrences and primary/ acquired resistances, as well as severe immune-related adverse events due to immunotherapeutic regimes. Therefore, more predictive preclinical assays are needed to further support preclinical and translational research, identifying novel druggable vulnerabilities to design more effective therapeutic strategies.

Material and Methods

Making use of our cohort of metastatic melanoma patient-derived xenografts (PDXs), we performed parallel in vivo and ex vivo drop-out genetic screens by means of a shRNA library targeting genes, for which drugs are already available and could be repurposed for melanoma treatment. We rationally combined targeted therapy agents to reach additive effect and we compare that to the standard-of-care treatment.

Results and Discussion

We proved that our ex vivo migration-based genetic screen is a feasible approach to isolate novel melanoma actionable vulnerabilities, predictive of drug efficacy. Our ex vivo system emerged as a reliable preclinical platform to define patients drug sensitivity profiles and identify alternative therapeutic strategies. In this setting, we proved that the dual targeting of Aurora kinase A (alisertib) and Mitogen-activated protein kinase MEK (trametinib) (AT) is highly effective in a wide panel of metastatic PDX, regardless their heterogeneous genetic background. Moreover, AT combination showed to outperformed the standard-of-care therapy (in BRAF-mutant PDXs) and to overcome resistance in therapy-resistant models. In two metastatic melanoma PDXs, AT combination dramatically inhibited in vivo tumor growth and significantly prolonged mice overall survival. AT revealed additive effect of pathways modulation, transcriptionally regulating a wider set of genes, compared to both monotherapies and unveiling novel points of intervention for a triple combination.

Conclusion

Ex vivo migration assay is a reliable tool to screen for drug sensitivity in highly translational valuable models. Alisertib trametinib combination is a promising alternative therapeutic approach that may be particularly beneficial for therapy-resistant patients.

Zocchi A.¹, Marocchi F.¹, Alfieri F.¹, Stinà S.², Mohammadi N.^1,3, Bresin A.⁴, Levati L.⁴, Russo G.⁴, D’Atri S.⁴, Lanfrancone L.¹

¹ Department of Experimental Oncology, European Institute of Oncology IRCCS, Milan, Italy

² Laboratorio di anatomia patologica, ASL AL, San Giacomo Hospital, Novi Ligure (AL), Italy

³ Experimental hematology, San Raffaele Institute, Milan, Italy

⁴ Laboratory of Molecular Oncology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy

Introduction

Clinical advances in melanoma treatment proved to be remarkable during the last decade. Introduction of systemic combination strategies targeted to specific mutations (BRAF and MEK inhibitors) and immune checkpoints (anti- PD1, CTLA4) dramatically improved overall survival rates. Nevertheless, a large fraction of BRAF-mutant patients develops primary or secondary resistances. Numerous therapeutic approaches were tested, many of them showing unbearable toxicities, mainly due to overlapping adverse events of the targeting agents. New combinations are now being tested, exploiting triple-agent combinations with immunotherapy, but with unsatisfactory results. Thus, the identification of novel vulnerabilities for drug resistant BRAF-mutant melanoma patients is a crucial clinical need.

Material and Methods

To this aim, we performed an shRNA-based in vivo genetic screen with a library that targets 195 actionable genes (FDAome library), each linked to specific FDA-approved available drugs that can be repurposed for melanoma treatment. A375 cells resistant to dabrafenib and trametinib (A375-DT) were used as a model to uncover new dependencies.

Results and Discussion

Our FDAome screen unveiled 37 genes which are essential to the acquisition of the DT-resistant phenotype. Six of these druggable genes showed overexpression in A375-DT cells compared to the parental cells and overexpression was confirmed also in another melanoma DT-resistant cell line (SKMEL28-DT). The validation of the selected hits is in progress. We will test the associated drugs as monotherapy or in combination with dabrafenib and trametinib, to assess their cytotoxic potential or their ability to re-sensitize cells to the standard-of-care therapy. The newly identified cytotoxic agents will be eventually combined with immunotherapy, to reach a more durable response. To this end we have generated a syngeneic mouse melanoma model. In parallel, we have established patient-derived primary cultures, some of which are resistant to DT. This model will be exploited to improve the translational potential of our findings.

Conclusion

To conclude, these approaches and models will allow us to develop new combinatorial treatments for relapsing melanoma patients by drug repurposing.

Stanislas du Manoir, Hélène Delpech, Béatrice Orsetti, William Jacot, Nelly Pirot, Pierre-Emmanuel Colombo, Claude Sardet, Charles Theillet

INSERM, Institut de Recherche en Cancérologie de Montpellier, France

Introduction

Most High-Grade Ovarian Carcinomas (HGOCs) are sensitive to carboplatin (CBP)-based chemotherapy but frequently recur within 24 months. Recurrent tumors remain CBP-sensitive and acquire resistance only after several treatment rounds. Recurrences arise from a small number of residual tumor cells hardly amenable to investigation in patients. We developed Patient-Derived Xenografts (PDXs) that allow the study of these different stages of CBP-sensitive recurrence and acquisition of resistance.

Material and Methods

We generated PDX models from CBP-sensitive and intrinsically resistant HGOC. PDXs were CBP- or mock-treated and tumors were sampled, after treatment and at recurrence. We also isolated models with acquired-resistance from CBP-sensitive PDXs. All tumors were characterized at the histological and transcriptome levels.

Results and Discussion

PDX models reproduced treatment response seen in the patients. CBP-sensitive residual tumors contained non-proliferating tumor cells clusters embedded in a fibrotic mesh. In non-treated PDX tumors and treated CBP-resistant tumors fibrotic tissue was not prevalent. Residual tumors had marked differences in gene expression when compared to naïve and recurrent tumors, indicating downregulation of cell cycle and proliferation and upregulation of interferon response and epithelial–mesenchymal transition. This gene expression pattern resembled that described in embryonal diapause and ‘drug-tolerant persister’ states. Residual and acquired-resistance tumors share the overexpression of three genes – CEACAM6, CRYAB, and SOX2.

Conclusion

In HGOC PDX, CBP-sensitive recurrences arise from a small population of quiescent, drug-tolerant, residual cells embedded in a fibrotic mesh. These cells overexpress CEACAM6, CRYAB and SOX2, a signature also associated with acquired resistance and poor patient prognosis, which, thus, might serve as a biomarker to predict recurrence and emergence of resistant disease in CBP-treated HGOC patients.

Carolina Velazquez¹, Esin Orhan¹, Imene Tabet¹, José Adélaide², Béatrice Orsetti¹, Lise Fenou¹, Daniel Birnbaum², Claude Sardet¹, William Jacot¹, Charles Theillet¹

¹ IRCM U1194 INSERM, Université de Montpellier, ICM, Montpellier France

² CRCM, INSERM, Université Aix-Marseille, IPC, Marseille, France

Introduction

TNBC is the most aggressive and the breast cancer subtype comprising the largest number of BRCA-deficient tumors. BRCA1 and BRCA2 are central genes to Homologous Recombination Repair (HRR) and tumors with HRR deficiency (HRD) show high sensitivity to platinum and PARP1 inhibitors. Hence, the detection of HRD is important in the clinical management of TNBC. Germline BRCA1/2 mutations have, until recently, been the only validated indications for PARPi therapy in breast and ovarian cancer, but data suggest that this could be extended to tumors with mutations and/or epigenetic silencing affecting other genes of the pathway. However, the actual sensitivity to PARPi of tumors with BRCA1-hypermethylation (15 to 20% of TNBC) is a disputed issue. Moreover, exposure of BRCA-deficient tumors to therapy frequently leads to resistance based on complete or partial restoration of HRR. Hence, the actual PARPi sensitivity of TNBC with HRD that have seen therapy prior to Olaparib treatment needs to be assessed.

Material and Methods

We interrogated the sensitivity to carboplatin (CBP) and to Olaparib (PARPi) of 7 TNBC and 1 High Grade Ovarian Serous Ovarian Carcinoma (HGSOC) PDX comprising 1 BRCA-proficient, 4 BRCA1 hypermethylated, 3 BRCA1-mutated models. Four (4) of the 7 TNBC PDX were established from tumors that had undergone neo-adjuvant chemotherapy (NACT) and 3/4 showed BRCA1 hypermethylation. Because definition of HRD on genetic bases does not preclude restoration of BRCA-proficiency, we used the functional assay based on the capacity of tumor cells submitted to a genetic insult to produce BRCA1 and RAD51 foci to infer the level of BRCA-deficiency.

Results and Discussion

Whereas, 5/7 BRCA1-deficient PDX showed at least 40% mean volume reduction under CBP, only 3/7 showed either stabilization or modest volume reduction with Olaparib. All other BRCA1-deficient models progressed under Olaparib treatment. Checking for BRCA1 protein expression by western blot we noted that all BRCA1 deficient models, except one PDX with heterozygous BRCA1 methylation, showed loss of expression of the full length BRCA1 protein. We assessed the formation of BRCA1 and RAD51 nuclear foci in PDX sampled at the end of Olaparib treatment. While 4/7 BRCA1-deficient models showed loss of BRCA1 foci formation, only 3 of the 4 BRCA1-foci-negative tumors formed RAD51 foci upon Olaparib treatment. The 3 RAD51-negative models had not seen NACT before grafting and were the only tumors showing partial response to Olaparib. All other PDX progressed under treatment and showed RAD51 foci.

Conclusion

We could confirm this association between strong reduction of RAD51 foci formation and the sensitivity to Olaparib in a series of BRCA1 proficient and deficient TNBC cell line models. Hence, BRCA1 and RAD51 foci formation could be a reliable test to be performed on TNBC patient biopsies to determine the level of BRCA-deficiency and orient the treatment.

Venkatasamy A.^1,2,3, Guerin E.¹, Reichardt W.³, Chenard MP.⁴, Romain B.^1,5, Gaiddon C.¹, Mellitzer G.¹

¹Streinth Lab (Stress Response and Innovative Therapies), Inserm UMR_S 1113 IRFAC, Interface Recherche Fondamental et Appliquée à la Cancérologie, 3 avenue Molière, Strasbourg, France

²IHU-Strasbourg, Institute of Image-Guided Surgery, Strasbourg, France

³Medizin Physik, Universitätsklinikum Freiburg, Kilianstr. 5a, 70106 Freiburg, Germany

⁴Pathology department, Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, 1 avenue Molière, 67098 Strasbourg CEDEX, France

⁵Digestive surgery department, Hôpital de Hautepierre, Hôpitaux Universitaires de Strasbourg, 1 avenue Molière, 67098 Strasbourg CEDEX, France

Introduction

Gastric cancer is an aggressive disease. Its poor prognosis can be explained by: i) late diagnosis due the lack of easy/affordable diagnostic tool, ii) resistances to treatments towards immunotherapy, due to a frequent low expression of immune check-point ligand PD-L1, or towards chemotherapy due to p53 mutations, and iii) comorbidity factors, notably muscle atrophy. To improve our understanding of this complex pathology and develop innovative diagnostic/treatment protocols, we established patient-derived xenograft (PDX) models and characterized the tumor ecosystem using a morpho-functional approach combining high-resolution imaging with molecular analyses, regarding the expression of biomarkers relevant to therapy and the presence of muscle atrophy.

Material and Methods

Human gastric cancer tissues samples (from patients) were implanted in nude mice. Established PDX, treated with cisplatin or not, were imaged by Magnetic Resonance Imaging (MRI) and analyzed using immunohistology, immunohistochemistry for relevant biomarkers (i.e., p53, PD-L1, PD-1, HER-2, CDX2, CAIX, CD31, -actin) and transcriptomics (RNAseq).

Results and Discussion

We obtained 5 different gastric adenocarcinomas PDX (success rate 21%): 3 well-differentiated, 1 moderately-differentiated and 1 poorly-differentiated adenocarcinomas. All models retained the architectural and histological features of their primary tumors across passages. MRI allowed in-real-time evaluation of differences between PDX, in terms of tumors substructure, post-therapeutic changes, including an insight on muscle changes and atrophy. Immunohistochemistry showed differential expression of p53, HER-2, CDX2, -actin, PD-L1, PD-1, CAIX and CD31 between models and upon treatment. In addition, transcriptomics showed that treatment induced hypoxia and metabolic reprograming in the tumor microenvironment.

Conclusion

Our PDX models are representative for different gastric cancer subtypes, reproducing quite well the heterogeneity and complexity of human tumors, with differences in structure, histology, muscle atrophy and the status for PD-L1/PD-1, p53, HER-2, and CDX2, which makes them valuable for the development of novel drugs and targeted therapies and analyze their impact on the tumor microenvironment.

Laia Monserrat¹, Claudia Yañez¹, Mónica Sánchez-Guixé², Cristina Viaplana³, Fara Brasó-Maristany^4,5, Meritxell Bellet^1,6, Sarat Chandarlapaty⁷, Lara Nonell⁸, Guillermo P Vicent⁹, Violeta Serra^1,10

¹ Experimental Therapeutics Group, Vall d’Hebron Institute of Oncology, Barcelona, Spain

² Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain

³ Oncology Data Science Group, Vall d'Hebron Institute of Oncology, Spain

⁴ Translational Genomics and Targeted Therapies in Solid Tumors, August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain

⁵ Department of Medical Oncology, Hospital Clínic of Barcelona, Spain

⁶ Breast Cancer and Melanoma Group, Vall d’Hebron Institute of Oncology, Barcelona, Spain

⁷ Departments of Pathology and Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, USA

⁸ Bioinformatics Unit, Vall d’Hebron Institute of Oncology, Barcelona, Spain

⁹ Molecular Biology Institute of Barcelona (IBMB-CSIC)

¹⁰ Vall d’Hebron Institute of Research

Introduction

Up to 90% of estrogen receptor-positive (ER+) breast cancers (BC) also express the androgen receptor (AR). Selective modulators of the AR (SARMs), such as RAD140, have shown clinical activity in endocrine therapy (ET)-resistance. However, the mechanism by which AR agonists block ER is not clearly understood. Moreover, the combination of a SARM plus CDK4/6-inhibitors (CDK4/6i) could be a novel therapeutic strategy for ER+/HER2- patients who do not tolerate ET. Here, we aimed to study the antitumor activity of RAD140 in a cohort of ER+/HER2- patient-derived xenografts (PDX) to identify biomarkers of response.

Material and Methods

Hormone-dependence was evaluated in 30 PDXs using ovariectomized (OVX) vs non OVX mice. Alterations in ESR1 and PIK3CA genes were determined with the MSK-IMPACTTM targeted exome panel and RNA-sequencing analysis. Pharmacodynamic experiments (PD) were conducted in 35 PDX models after 5-days treatment with the selective ER degrader fulvestrant, RAD140 or dihydrotestosterone (DHT). Protein levels of AR, ER and Ki67 were assessed by immunohistochemistry. Modulation of cell-cycle proteins by AR agonists was evaluated by Western blot. In vivo antitumor activity of RAD140 alone or in combination with palbociclib was tested in comparison to fulvestrant plus palbociclib.

Results and Discussion

Ten out of thirty ER+/HER2- PDXs harbored ESR1-mutations or fusions and were hormone-independent in vivo. PD experiments showed a Ki67-antiproliferative response with both RAD140 and DHT in 10 out of 35 models. We confirmed sensitivity to RAD140 monotherapy in 4 out of 7 Ki67-responsive models tested. Interestingly, AR agonists decreased E2F1, cyclin A/E2 protein levels in RAD140-sensitive but not resistant-PDX. RAD140 showed equal antitumor activity than fulvestrant when combined with palbociclib in 7 out of 8 models tested and superior activity in an ESR1-mutant model.

Conclusion

RAD140 exhibits antitumor activity in hormone-independent and ESR1-altered ER+/HER2- PDX models as single agent or in combination with CDK4/6i. Mechanistically, AR agonists decrease proliferation as shown by a decrease in Ki67 and S/G2-cell cycle cyclin levels.

Magdalena Cybulska¹, Kamila Kruczkowska-Tarantanowicz², Anna Polak³, Katarzyna Kyc-Wachowiak⁴, Urszula Pakulska⁵, Tomasz Rzymski⁵, Jolanta Korsak⁴, Przemysław Juszczynski³, Piotr Rzepecki², Michal Mikula¹

¹ Department of Genetics, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland

² Department of Internal Medicine and Hematology, Military Institute of Medicine, Warsaw, Poland

³ Institute of Hematology and Transfusion Medicine, Department of Experimental Hematology, Warsaw, Poland

⁴ Department of Clinical Transfusiology, Military Institute of Medicine, Warsaw, Poland

⁵ Biology R&D, Ryvu Therapeutics S.A., Krakow, Poland

Introduction

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcomes. Preclinical model systems are required to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow the development of AML models for both in vivo and ex vivo functional studies with patient-derived AML cells. RVU120 (SEL120) is a specific, selective inhibitor of CDK8 and its paralog CDK19, and preclinical data indicate high efficacy of RVU120 in AML models, particularly in cells with stem cell-like characteristics. Here we tested RVU120 efficacy on our AML PDX models in vivo alone and ex vivo alone and combination with venetoclax.

Material and Methods

NSG-SGM3 mice received sublethal total body irradiation (2 Gy), the next day mice were injected intravenously with primary AML blast from donors. Engraftment and tumor burden was followed up in the peripheral blood monthly. For PDX model propagation a range of 1–2 million unsorted human CD45+ CD33+ cells was transferred into NSG-SGM3 mice via intravenous injection. RVU120 was administrated orally at 45 mg/kg once a day. For ex vivo treatment PDX were cultured in S7 media and treated with RVU120 alone and in combination with venetoclax.

Results and Discussion

Using the NSG-SGM3 mouse strain we have established a platform of ten AML PDX models that have served to show the preclinical efficacy of RVU120. In vivo studies indicate the strong antileukemic potential of RVU120 that was associated with the multilineage commitment of CD34+ AML cells. Ex vivo experiments showed a synergistic effect of RVU120 and venetoclax in CD34+ AML cells.

Conclusion

We have established a first-in-Poland AML PDX platform for preclinical evaluation of new therapies that is exemplified by the RVU120 preclinical efficacy that allowed its transition to clinical development phases.

Jorge Gómez-Miragaya¹, Bogdan-Tiberius Preca¹, Maren Diepenbruck¹, Marta Palafox¹, Marie May Coisseux¹, Marcus Vetter², Christian Kurzeder³, Walter Weber⁴, and Mohamed Bentires-Alj¹

¹ Department of Biomedicine, University of Basel, Basel, Switzerland;

² Gynecological Cancer Centre, Hospital for Women, University of Basel, Basel, Switzerland;

³ SAKK & University Hospital of Basel, Basel, Switzerland;

⁴ Breast Center, University Hospital Basel, Basel, Switzerland.

Introduction

Hormone receptor positive (HR+) breast cancer represents 70% of all cases and is the most prevalent form of metastatic breast cancer, the main breast cancer-related cause of death. Several studies have identified the relevance of the cyclin-D1/CDK4/6/RB1 pathway in HR+/HER2- breast cancer. The combination of CDK4/6 inhibitors and endocrine therapy significantly increase progression free-survival and overall survival compared to endocrine therapy alone in patients with HR+/HER2- advanced breast cancer. This fact led to their approval by the FDA as first line for the treatment treatment of patients with HR+/HER2- advanced breast cancer, in combination with endocrine therapy. Nevertheless, after a period of response, treatment resistant tumors emerge, and few therapeutic options are available at this stage. Mechanisms underlying the emergence of resistance to CDK4/6 inhibitors are poorly understood and their clinical relevance has not been clearly established. Therefore, there is an urgent need to identify these mechanisms and develop new therapies to overcome resistance.

Material and Methods

In our study, we initially evaluated the response of HR+/HER2- breast cancer patient-derived xenograft (PDX) models to endocrine therapy (i.e., fulvestrant), CDK4/6 inhibitors (i.e., ribociclib) and the combination. To study biological dynamics during treatment and emergence of resistance, transcriptomics, proteomics and bioinformatics analyses comparing matched sensitive and resistant HR+/HER2- breast cancer PDX models are ongoing. We will characterize and perform functional assays in resistant tumors, and identify essential targetable pathways to overcome resistance.

Results and Discussion

Mimicking clinical scenario, HR+/HER2- breast cancer PDX models that were initially sensitive to fulvestrant and fulvestrant plus ribociclib progressively acquired resistance upon continuous in vivo drug administration. Drug-resistant lung metastases were found in two out of the three drug-resistant PDX models. This study will pinpoint novel targets for second line therapy and should improve clinical management of patients with HR+/HER2- advanced breast cancer.

Conclusion

All ER+ breast cancer PDX models tested are initially resistant to ribociclib as single agent.

42% of the HR+/HER2- breast cancer PDX models tested show a statistically significant benefit for the combination compared with the single agent fulvestrant. After continuous in vivo drug exposure HR+/HER2- BC PDX models develop resistance to fulvestrant and fulvestrant plus ribociclib. Breast cancer-derived lung metastases are found in all treatment arms from two different HR+/HER2- breast cancer PDX models.

Tom Van Nyen^1,2, Mélanie Planque^3,4, Lara Rizzotto⁵, Wout De Wispelaere¹, Hugo M. Horlings⁶, Ben Davidson⁷, Reuven Agami², Sarah-Maria Fendt^3,4, Daniela Annibali^1,2 and Frédéric Amant^1,8,9

¹ Gynecological Oncology Laboratory, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), 3000 Leuven, Belgium

² Division of Oncogenomics, Oncode Institute, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands

³ Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer Biology, VIB, Herestraat 49, 3000 Leuven, Belgium

⁴ Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), Herestraat 49, 3000 Leuven, Belgium

⁵ TRACE PDX Platform, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), 3000 Leuven, Belgium

⁶ Division of Molecular Pathology, The Netherlands Cancer Institute, 1066CX, Amsterdam, the Netherlands

⁷ University of Oslo, Faculty of Medicine, Institute of Clinical Medicine, N-0316 Oslo, Norway; Department of Pathology, Oslo University Hospital, Norwegian Radium Hospital, N-0310 Oslo, Norway

⁸ Department of Obstetrics and Gynecology, University Hospitals Leuven and Department of Oncology, 3000 Leuven, Belgium

⁹ Centre for Gynecologic Oncology Amsterdam (CGOA), Antoni Van Leeuwenhoek-Netherlands Cancer Institute (AvL-NKI), University Medical Center (UMC), Amsterdam, The Netherlands

Introduction

Resistance to platinum-based chemotherapy represents a major clinical and societal challenge for the management of many cancer patients, particularly those with epithelial ovarian cancer. Although the majority initially respond, a high proportion experiences several response-relapse-response events during the course of the disease, until tumors become resistant. Despite improved knowledge of the molecular determinants of platinum resistance, lack of clinical applicability limits exploitation of many potential targets, leaving patients with limited options. Serine biosynthesis has been linked to cancer growth and poor prognosis in various cancer types, however its role in platinum-resistant ovarian cancer has not been investigated before.

Material and Methods

Phosphoglycerate dehydrogenase (PHGDH) expression has been determined in matched biopsies from ovarian cancer patients collected longitudinally at diagnosis and after platinum treatment. Metabolomic and molecular analyses have been performed on the isogenic ovarian cancer cell lines A2780 (wt/cis). Results have been confirmed in sensitive, resistant and matched ovarian cancer PDX models and PDX-derived organoid cultures.

Results and Discussion

We found that a subgroup of resistant tumors decreases phosphoglycerate dehydrogenase (PHGDH) expression at relapse after platinum-based chemotherapy. Mechanistically, we observe that this phenomenon is accompanied by a specific oxidized nicotinamide adenine dinucleotide (NAD+) regenerating phenotype, which helps tumor cells in sustaining Poly (ADP-ribose) polymerase (PARP) activity under platinum treatment. Consequently, combining carboplatin and PARP or NAD+ synthesis inhibitors affects the growth of resistant models with decreased serine synthesis activity.

Conclusion

Our findings have strong potential clinical applicability, because they identify alterations in serine and NAD+ metabolism as actionable vulnerabilities in a subgroup of platinum resistant ovarian cancers, and provide a rationale to test novel combinatorial therapeutic approaches to target resistance.

Robert E. Hynds¹, Ariana Huebner¹, David R. Pearce¹, Oriol Pich², Ayse U. Akarca¹, Sophia Ward², David A. Moore¹, Teresa Marafioti¹, Nicholas McGranahan¹, Charles Swanton²

¹ University College London, London, UK

² The Francis Crick Institute, London, UK

Introduction

Patient-derived xenograft (PDX) models have become important models in cancer biology. They are thought to mimic tumor biology more closely than traditional cell lines as a consequence of their in vivo heterocellularity and cell-matrix interactions, their 3D architecture and their relatively recent derivation. The genomic fidelity of PDXs is integral to their applications in both basic research and in translational medicine.

Material and Methods

We derived multi-region PDX models from patients enrolled in TRACERx – a study of the evolutionary dynamics of non-small cell lung cancer (NSCLC) that uses a multi-region deep whole-exome sequencing (WES) approach – using more extensive spatial sampling to better understand the histological and genomic fidelity of the PDX approach.

Results and Discussion

We transplanted regional NSCLC tumor tissue subcutaneously into immunocompromised NSG mice. 134 regions were attempted, resulting in 60 passagable xenografts (44.8%). Of these, 44 regional xenografts (32.8% of all regions) were NSCLC-derived, while 16 (11.9% of all regions) were B-cell lymphoproliferative disease. Multi-region sampling revealed heterogeneous success of PDX derivation between regions within individual patient tumors. There was correlation between PDX derivation and tumor purity by WES. Overall, NSCLC PDX models were established for 22 patients (50%; range 1-5 regional models per patient). Histologically, broad similarity was observed between PDX models and corresponding patient tumor regions, although in a small number of models, variability was noted between the patient tumor and the corresponding PDX model, or during PDX passaging. PDXs were subjected to WES for comparison with matched patient tumor regions.

Conclusion

Our study demonstrates the feasibility of systematic multi-region PDX derivation and suggests that multiple spatial sampling of tumors could improve PDX take rates and generate PDXs that represent the intratumor diversity of heterogenous NSCLCs.

Annelies Van Hemelryk^1,#, Ingrid Tomljanovic^1,2, Corrina M.A. de Ridder¹, Debra C. Stuurman¹, Wilma J. Teubel¹, Martin E. van Royen³, Harmen J.G. van de Werken^1,4,5, Magda Grudniewska², Guido W. Jenster¹, Wytske M. van Weerden¹

¹ Department of Urology, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

² GenomeScan B.V., Leiden, The Netherlands

³ Department of Pathology, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

⁴ Cancer Computational Biology Center, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

⁵ Department of Immunology, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

^# These authors contributed equally

Introduction

Castration-resistant prostate cancer (CRPC) is a highly heterogeneous and lethal form of advanced prostate cancer (PCa), that remains reliant on a reactivated and often highly augmented AR signaling. Improving therapeutic strategies is crucial to maximize patient survival, but requires comprehensive testing on robust and representative preclinical models. Patient-derived xenografts (PDXs) from CRPC patients on contemporary therapies are scarce and provide an incomplete representation of CRPC heterogeneity. Complementing our existing PDX panel, we now introduce a new set of five AR-positive (AR+) PDXs and matching PDX-derived organoids (PDXOs) obtained from heavily pretreated CRPC patients.

Material and Methods

A total of 38 PCa tumor samples were implanted subcutaneously into intact male mice. In parallel, we developed a workflow to create organoids from PCa xenografts. PDXs and PDXOs were extensively characterized by means of histopathology, RNA sequencing and microarray analysis. Genomic and transcriptomic profiles were compared to original tumors and independent cohorts of CRPC patients. AR signaling status was evaluated using expression levels of genes defining the pathway and effect of bilateral orchiectomy was assessed in PDXs, while PDXOs were exposed to anti-androgen treatment (enzalutamide). Responses of PDXs and PDXOs to taxane treatment (docetaxel and cabazitaxel) were evaluated and retrospectively compared to matching patient responses.

Results and Discussion

We developed five new CRPC PDXs, two of which were derived from the same patient at different disease stages, and generated matching PDXOs. All are high-grade AR+ adenocarcinomas that express PSA, with one PDX-PDXO pair displaying a cribriform growth pattern mixed with ductal features, all reflecting donor patient tumors. Concordance of transcriptomic profiles between native tumors, PDXs and PDXOs further validated model consistency. Across PDXs, we confirmed the presence of common PCa-associated genomic alterations, including losses and gains of TP53, PTEN, RB1 and MYC. TMPRSS2-ERG gene fusion was identified in one and AR amplification in four CRPC PDXs. The continued reliance on AR signaling was preserved across the entire set and we observed tumor regression upon surgical castration followed by tumor regrowth after reintroducing testosterone. PDXs and PDXOs accurately reflected matching patient responses to anti-androgen and taxane treatment, providing perspectives for individualized drug response profiling and patient stratification.

Conclusion

This new set of CRPC PDXs and matching PDXOs, that faithfully reproduces key characteristics and behavior of CRPC patient tumors, constitutes a reliable and unlimited resource for in-depth studies of treatment-induced, AR-driven resistance mechanisms and for tumor-specific drug testing in late stage castration-resistant disease.

Annelies Van Hemelryk¹, Sigrun Erkens-Schulze¹, Lifani Lim¹, Corrina M. A. de Ridder¹, Debra C. Stuurman¹, Pim J. French^2,3, Martin E. van Royen⁴, Wytske M. van Weerden¹

¹ Department of Urology, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

² Cancer Treatment Screening Facility, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

³ Department of Neurology, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

⁴ Department of Pathology, Erasmus MC Cancer Institute, University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands

Introduction

Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, standardized drug testing procedures are generally lacking, introducing experimental variability that complicates reproducibility, both within and between institutions. Moreover, common viability assays are restricted to endpoint measurements and viability as the sole read-out, losing important information on key biological aspects that might be exclusive to 3D organoids and that may be impacted by the administered drugs. In this study, we aimed to standardize a viability-based drug testing procedure for organoids derived from PCa patient-derived xenografts (PDXs). In addition, we developed and optimized an image-based live/dead assay using high-content fluorescence live-cell imaging in living PCa organoids.

Material and Methods

To initiate PCa PDX-derived organoids (PDXOs), PDX tumors were dissected and digested enzymatically, passed through a 100 µm cell strainer and resuspended in a synthetic, thermo-sensitive, hydrogel. Organoids were allowed to assemble in 24-well plates and harvested after 7 to 14 days by dissolving the hydrogel dome. Organoids were plated at a density of 5000 – 10000 organoids per 8 µL hydrogel domes in 96-well plates. Organoids were incubated with dose ranges of standard PCa chemotherapeutics (docetaxel and cabazitaxel) and of the anti-androgen enzalutamide. Organoid viability was measured with CellTiter-Glo (CTG) 3D (Promega). An optimized fluorescent dye combination of caspase 3/7, propidium iodide and Hoechst 33342 was used for imaging experiments. Confocal time-lapse imaging was performed with the Opera Phenix High Content Screening System (PerkinElmer). Staurosporine treatment was used as positive control, solvents as negative controls in both CTG assays and image-based drug tests.

Results and Discussion

We developed a standardized protocol for performing CTG-based drug tests in PCa PDXOs and found 3 factors to be crucial for reliable interpretation of viability assays in these PDXOs: presence of ROCK-inhibitor in organoid medium, organoid proliferation rate and treatment duration. Moreover, we generated an image-based live/dead assay and implemented a custom image analysis method to quantify the number of organoids, organoid sizes and the number of apoptotic, necrotic and viable cells. This image-based procedure provides valuable insights on treatment effects in living organoids.

Conclusion

We developed two protocols for PCa PDXO-based drug testing and provide additional read-outs with our image-based procedure. Both procedures might well be customized for PDX- and patient-derived organoids from various cancer types.

Adria Bernat-Peguera, Juan Navarro-Ventura, Laura Lorenzo-Sanz, Victoria da Silva-Diz, Luis Palomero, Rosa M. Penin, Miren Taberna, Eva Gonzalez-Suarez, Alberto Villanueva, Purificación Muñoz

IDIBELL, L'Hospitalet de Llobregat, Spain

Introduction

Recurrent and/or metastatic unresectable cutaneous squamous cell carcinomas (cSCCs) are treated with chemotherapyor radiotherapy but have poor clinical responses. A limited response (up to 45% of cases) to EGFR-targeted therapies was observed in clinical trials with patients with advanced and metastatic cSCC. Here, we analyze the molecular traits underlying the response to EGFR inhibitors, and the mechanisms responsible for cSCC resistance to EGFR-targeted therapy.

Material and Methods

We generated primary cell cultures and patient cSCC–derived xenografts (cSCC-PDXs) that recapitulate

the histopathologic and molecular features of patient tumors. Response to gefitinib treatment was tested and gefitinib-resistant (GefR) cSCC-PDXs were developed. RNA sequence analysis was performed in matched untreated and GefR cSCC-PDXs to determine the mechanisms driving gefitinib resistance.

Results and Discussion

cSCCs conserving epithelial traits exhibited strong activation of EGFR signaling, which promoted tumor cell proliferation, in contrast to mesenchymal-like cSCCs. Gefitinib treatment strongly blocked epithelial-like cSCC-PDX growth in the absence of EGFR and RAS mutations, whereas tumors carrying the E545K PIK3CA activating

mutation were resistant to treatment. A subset of initially responding tumors acquired resistance after long-term treatment, which was induced by the bypass from EGFR to FGFR signaling to allow tumor cell proliferation and survival upon gefitinib treatment. Pharmacologic inhibition of FGFR signaling overcame resistance to

EGFR inhibitor, even in PIK3CA-mutated tumors.

Conclusion

EGFR-targeted therapy may be appropriate for treating many epithelial-like cSCCs without PIK3CA-activating mutations. Combined EGFR- and FGFR-targeted therapy may be used to treat cSCCs that show intrinsic or acquired resistance to EGFR inhibitors.

Jara Garcia, Kuan Yan, Sander Basten, Leo Price, Bram Herpers

CrownBio, Leiden, the Netherlands

Patient-derived organoids (PDOs) are established by stimulating the maintenance and proliferation of (cancer) stem cells with WNT pathway activators and receptor tyrosine kinase (RTK) agonists in an extracellular matrix environment. Here we describe a large-scale image-based screen with dual-targeting (RTKxWNT) bispecific antibodies in a diverse panel or (metastatic) colorectal cancer organoids and (matched) normal organoid models. Out of more than 500 antibodies a potent EGFRxLGR5 bispecific antibody, MCLA-158, was identified. The antibody inhibited the outgrowth of a large number of different CRC PDO models, including KRAS mutant models, but shows minimal toxicity towards healthy colon organoids. Functionally, the antibody blocks RTK signaling by triggering EGFR degradation in an LGR5-dependent manner, and was shown to block metastasis initiation and to be able to suppress the outgrowth of in vivo tumor models of several other cancer types. This study highlights Crown Bioscience’s application of organoids throughout the drug discovery process – from high throughput screening to patient stratification and mechanism of action studies.

Olivier Déas, Léa Sinayen Emilie Indersie, Sophie Banis, Kathleen Flosseau, Enora Le Ven, Jean-Gabriel Judde, Stefano Cairo

XenTech, Evry, France

Introduction

Although Patient-derived xenografts (PDXs) are indispensable for preclinical studies, they suffer from limitations due to study costs related to tumor maintenance on mice, variable engraftment rate, growth delay and limited throughput for large-scale drug screening. To address this problem, we developed a panel of PDX-derived low-passage cell lines as a time and cost-effective in vitro pre-screening platform to profile compound activity.

Material and Methods

PDX-derived cell cultures were obtained from tumors explanted from mice and isolated by mechanical and enzymatic dissociation and maintained in culture at 37°C in a humidified atmosphere with 5% CO2 in complete growth medium. Cells are maintained in culture in XT-modified Advanced medium with 8% FBS with or without Rho-associated kinase (ROCK) inhibitor supplementation. For ATP measurement (CellTiter Glo® assay kit), cells were seeded in 96-well plates at a density of 1.25e3 to 5.10e3 cells/well. Cells were incubated 48h at 37°C prior to the addition of test molecules or vehicle at desired final concentrations. For PARP inhibition assays, cells were seeded in 6-well plates at a density of 10e5 to 5.10e5 cells/well. Cells were incubated up to 3 days prior to addition of olaparib (10 µM) at D0. At D4, D11 and D18, the medium was renewed with olaparib renewal. At D0, D7, D14 and D21, cells were stained with crystal violet (0.1% crystal violet, 2% ethanol) for 20 min and washed with water.

Results and Discussion

PDXs from several entities including breast, lung, colon, prostate, melanoma, glioblastoma and hepatoblastoma were tested for their capacity to generate cell lines maintaining the characteristics of the parental PDX tumor and usable for in vitro assays. Tumor cells isolated from PDX tumor tissue were cultured under different media and matrix conditions, allowing at least 5 passages in culture. Starting from a series of 60 PDX models, we developed 52 PDX-derived cell lines (86% success rate). A Short Tandem Repeat (STR) comparison profile was done with the parental PDX before performing a master bank to ensure cells’ origin, then whole exome sequencing (WES) and RNA sequencing (RNA-seq) were done for all models. Short term 2D cytotoxicity assays and long-term colony assays to compare cell lines in vitro drug sensitivity with the parental PDX drug response in vivo. We tested PARP inhibitors, an ADC (T-DM1) and an EGFR-targeting therapy (osimertinib).

Conclusion

PDX-derived cell lines tested as 2D cultures with drug-tailored experimental design mimic drug sensitivity observed in the parental PDXs in vivo. This in vitro platform could be used to perform preliminary drug screenings to select candidates to be validated in vivo on the parental PDX models.

Alex Martínez-Sabadell^{1,2 #}, Macarena Román¹, Irene Rius Ruiz^1,2, Beatriz Morancho¹, Marta Escorihuela¹, Christian Klein³, Marina Bacac³, Joaquín Arribas^{1, 2, 5, 6, 7,*}, Enrique J. Arenas^{1, 2, #,*}

¹ Preclinical Research Program and ² CIBERONC, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, 08035, Spain.

³ Roche Innovation Center Zurich, Roche Pharma Research & Early Development, pRED, Zurich, Switzerland.

⁴ Department of Biochemistry and Molecular Biology, Universitat Autónoma de Barcelona, Campus de la UAB, 08193, Bellaterra, Spain.

⁵ Cancer Research Program, IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain.

⁶ Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010, Barcelona, Spain.

^# These authors contributed equally

^* These authors jointly supervised this work

Introduction

Elimination of cancer cells by effector immune cells represents the culmination of a complex cascade of events, and disruption of any of those events may result in resistance. T cell-engaging therapies, such as T cell bispecific antibodies (TCBs) or chimeric antigen receptors (CARs), are raising extraordinary expectations as future treatments for virtually all cancers. Encouraging these expectations, TCBs and CARs have been recently approved to treat some hematologic malignancies. In contrast, TCBs and CARs against solid tumors tested to date, have failed to show clinical efficacy. This failure prompted intense research and the subsequent identification of mechanisms of primary and acquired resistance. All these mechanisms impinge on the ability of T cells to reach cancer cells and/or on the inhibition of T cells. However, little is known about putative intrinsic mechanisms of resistance of cancer cells. That is, mechanisms deployed by tumor cells to resist killing by fully active and correctly engaged T cells.

Objective

To identify novel mechanisms of acquired resistance to T cell redirection therapies and how to overcome it.

Material and Methods

Using HER2+ cell lines and patient-derived xenografts (PDXs) and a TCB targeting HER2, we generated in vitro and in vivo acquired resistant models to HER2-TCB. This resistance was obtained after coculturing in vitro with peripheral blood mononuclear cells (PBMCs) or in vivo in fully humanized mice with CD34+ cells obtained from cord blood, both from healthy donors.

Results and Discussion

The Here we demonstrate that disruption in interferon-gamma (IFN-gamma) signaling in cancer cells is a mechanism of intrinsic resistance to killing by fully active, correctly engaged, T lymphocytes. Importantly, we have identified that the kinase JAK2, which transduces the signal initiated by IFN-gamma, is the component preferably disrupted to acquired resistance across all models used.

Conclusion

These results unveil a novel mechanism of resistance to T cell based therapies, and imply the potential use of JAK2 and IFN-gamma response as a surrogate biomarker of response to immunotherapies. In addition, they open the avenue for the screening for therapies that can overcome deficient interferon-gamma response or restore JAK2 levels, which are promising potential candidates to increase the benefits of immunotherapies. Currently we are identifying novel genes and drugs in order to overcome resistance to immunotherapies by using a genome wide CRISPR screening and a drug screening, respectively, which will allow to characterize in depth a mechanism of resistance to redirected lymphocytes and will allow to design novel and more efficacious immunotherapy-based strategies for the treatment of different cancers.

Marta Palafox¹, Jorge Gómez-Miragaya¹, Tiberius Preca¹, Maren Diepenbruck¹, Simone Muenst², Daiana Stolz³, Marcus Vetter⁴, Christian Kurzeder⁵, Walter Weber⁶ and Mohamed Bentires-Alj¹

¹ Department of Biomedicine, University of Basel, Basel, Switzerland

² Institute of Pathology and medical Genetics, University Hospital Basel, University of Basel, Basel, Switzerland

³ Clinic of Respiratory Medicine and Pulmonary Cell Research, University Hospital Basel, Basel, Switzerland

4Gynecological Cancer Centre, Hospital for Woman, University of Basel, Basel, Switzerland

⁵ SAKK & University Hospital of Basel, Basel, Switzerland

⁶ Breast Center, University Hospital of Basel, Basel, Switzerland

Introduction 

Patient-derived xenograft (PDX) tumors are relevant preclinical models to study the behavior and heterogeneity of breast cancer (BC) as well as to identify predictive biomarkers and mechanisms of resistance to antitumor drugs, providing a powerful tool for clinical oncology.

Material and Methods

BC patient samples freshly resected from primary tumors (PT) or metastasis (MET) were collected for PDXs generation in NSG mice. Tumor pieces from triple negative (TN), HER2+ or unknown BC intrinsic subtypes were orthotopically implanted into the mammary fat pat (MFP). The hormone receptor positive (HR+) samples were engrafted into the MFP or single cells were injected intraductally into the mammary gland (MINDly) and supplemented with estradiol (8µg/ml) in the drinking water. The number of cells injected MINDly for giving rise to a PDX was assessed. PDXs generated were serially transplanted into the MFP of new NSG mice. The percentage of human (hCD298+) and lymphocytic (hCD45+) cell in PDXs were analyzed by FACS and the expression of relevant histopathological markers (ER, PR, HER2 and Ki-67) was quantified by immunohistochemistry (IHC). The response to fulvestrant (5 mg/mouse) and estradiol (E2) dependency were tested in 2 HR+ BC PDXs.

Results and Discussion

From 2016 to 2020, 69 samples from TN (n=18; 26%), HR+/HER2- (n=35; 51%), HER2+ (n=8; 11.5%) or unknown (n=8; 11.5%) BC subtypes were collected from PT (n=46; 67%) or different MET (n=23; 33%) and implanted in NSG mice. Twenty-four PDXs grew in passage 1 (35%) and 13 were established in passage 3 (19%), 12 of them coming from PT (26%) and one from a brain MET (4%). Three out of 13 PDXs were CD298- (23%) and one was hCD45+ (8%), which were discarded for subsequent analysis. The IHC showed that 5 PDXs were TN (28%), 3 HR+/HER2- (9%) and one HER2+ (13%), all of them resembling the intrinsic subtype of the initial BC sample. Regarding the HR+/HER2- PDXs, 7 out of 21 (33%) tumors that were implanted into the MFP and 2 out of 12 (17%) tumors that were implanted MINDly gave rise to a tumor in passage 1. The number of cells injected MINDly with the highest success rate (100%) was 5x105 cells/mammary gland, although the injection of 2.5x105 or 8x104 cells/mammary also generated tumors but less efficiently (25% and 50%, respectively). Fulvestrant did not show antitumor activity in UHB101 PDX (low expression of ER) whereas UHB204 (high expression of ER) was sensitive to the treatment, and its growth was independent of E2.

Conclusion

In summary, we generated a collection of 9 BC PDXs, which correspond to the BC intrinsic subtypes in patients. The engraftment ability of TNBC tumors was higher than HR+/HER2- and HER2+, but it was independent to the tissue of origin (PT/MET) or the method of implantation (MFP/MINDly) in the HR+/HER2- tumors. Finally, we demonstrated that our PDXs recapitulate the antitumor activity of endocrine treatments employed in the clinics.

Anaïs Oudin^1*, Pilar M. Moreno-Sanchez^1,2*, Yahaya A. Yabo^1,2, Eliane Klein¹, Virginie Baus¹, Aurélie Poli³, Simone P. Niclou¹, Anna Golebiewska¹

¹ NORLUX Neuro-Oncology Laboratory, Department of Cancer Research, Luxembourg Institute of Health, Luxembourg, Luxembourg;

² Faculty of Science, Technology and Medicine, University of Luxembourg, L-4367 Belvaux, Luxembourg;

³ Neuro-Immunology Group, Department of Cancer Research, Luxembourg Institute of Health, L-1526 Luxembourg, Luxembourg

Introduction

Glioblastoma (GBM) is the most deadly primary brain tumor despite a very aggressive treatment, which consists of surgical resection followed by radiotherapy and chemotherapy. Most GBM clinical trials fail due to inappropriate selection of the compounds at the preclinical stage. Therefore, appropriate preclinical models are crucial for achieving better treatment outcomes. GBM patient-derived othotopic xenografts (PDOXs) faithfully recapitulate the molecular and histopathological profiles of patient tumors. Orthotopic implantation allows for analogous tumor microenvironment and drug distribution though the blood brain barrier. However, current xenograft models suffer from the reduction of immune system components, which represents a bottleneck for adequate immunotherapy testing. Recapitulation of the standard-of-care regimen is challenging and it remains to be seen whether procedures combining surgical resections with clinically-relevant treatment protocols are practicable.

Material and Methods

We regularly derive GBM PDOXs by intracortical implantation of primary patient-derived organoids in immunodeficient mice (NSG, Nude). To introduce the human adaptive immune system, GBM organoids were implanted in the brains of the human CD34+ hematopoietic stem cell-engrafted NSG (HU-CD34+) mice. Tumor growth was followed by Magnetic Resonance Imaging. Detailed characterization of the tumor microenvironment was performed by single cell transcriptomics, immunohistochemistry and multicolor flow cytometry. To mimic tumor recurrence, we performed surgery and tumor resection at the early tumor development stage. Robust in vivo treatment protocols were established for standard-of-care, novel drugs and combinatory treatments.

Results and Discussion

We show that PDOX models recapitulate major components of the tumor microenvironment found in human GBM, including microglia, astrocytes, oligodendrocytes and endothelial cells. Mouse cells reciprocally crosstalk with human GBM cells and switch towards GBM-instructed phenotypes. Missing adaptive immune system can be introduced by tumor growth in HU-CD34+ mice. HU-CD34+ mice present more than 50% of human immune cells in the bone marrow and blood. We observed an influx of human immune cells to the tumors developed in the brain. We further show that surgical resection can be applied on mouse brain and GBM tumors regrow after surgery similarly to patient GBM, leading to delayed survival time. Magnetic Resonance Imaging allows for robust assessment of the tumor growth and drug responses in vivo.

Conclusion

Our data provide insights into the recapitulation of the heterogeneous tumor microenvironment instructed by GBM cells in PDOXs. This work further confirms the wide applicability of GBM PDOXs for recapitulation of standard-of-care protocols and testing of novel treatments including immunotherapeutics.

Lubomir Minarik^1,2, Kristyna Pimkova¹, Juraj Kokavec¹, Frederic Vellieux¹, Vojtech Kulvait¹, Karina Savvulidi Vargova³, Petra Kralova Viziova⁴, Radislav Sedlacek⁴, Zuzana Zemanova⁵ and Tomas Stopka

¹ BIOCEV, 1st Medical Faculty, Charles University, 25250 Vestec, Czech Republic;

² Clinic Haematology, General Faculty Hospital, 12808 Prague, Czech Republic

³ Pathophysiology, 1st Medical Faculty, Charles University, 12853 Prague, Czech Republic;

⁴ Czech Centre for Phenogenomics, Institute of Molecular Genetics, 25250 Vestec, Czech Republic;

⁵ Cytogenetics, General Faculty Hospital, 12808 Prague, Czech Republic

Introduction

The mechanisms by which myelodysplastic syndrome (MDS) cells resist the effects of hypomethylating agents (HMA) are currently the subject of intensive research. A better understanding of how MDS cells bypass a progressive decrease in efficacy of HMA and progress to acute myeloid leukemia (AML) requires the development of new cellular models.

Material and Methods

We utilized MDS/AML cell lines and developed model of 5-azacytidine (AZA) resistance whose stability was validated by an orthotopic intra femoral transplantation into NSGS mice. Proliferation of cells has been monitored weekly with luminescence using Spectral Lago X instruments Imaging, images were processed with Aura Imaging Software. Mice were transplanted either with azacitidine sensitive (AZA-S) or resistant (AZA-R) cells and treated with AZA 3 times a week until progression of leukemia. AZA-R evolution was monitored using whole exome sequencing (WES), cytogenetics, and RNAseq.

Results and Discussion

By integrated analysis of expression and mutations, we observed deregulated phosphatidylinositol 3 kinase (PI3K)/AKT signaling, RAF/MEK/ERK signaling, SRC signaling, and nuclear processes involving HDAC and BRD4. We further showed that these pathways can be modulated by specific inhibitors that significantly block the proliferation of AZA-R cells but are unable to increase their sensitivity to AZA. Our data reveal a set of molecular mechanisms of the AZA-R phenotype, which can be used for broadening therapeutic options at progressing states during AZA therapy. In the matter of increasing sensitivity of AZA-R cells to AZA, we observed aberrant changes in cellular redox homeostasis that can be targeted using specific inhibitors involved in ROS production.

Conclusion

Our work provides new insights into the mechanisms of AZA resistance phenotype and targetable pathways that that can be further explored in mouse models in vivo in order to find new therapeutic strategies. Engraftment of AZA-R cells into NSGS mice resulted in significantly shorter overall survival and lower bioluminescent signal in comparison with AZA-S cells.